The goal of this research is to elucidate the molecular mechanisms involved in regulation of transcription, using as model systems two catabolite sensitive operons of E. coli. A variety of biophysical and biochemical methods will be applied to study interactions of the catabolite activator protein (CAP) and RNA polymerase with DNA fragments containing wild type or mutant promoter regions of the lactose and galactose operons. The techniques to be used include gel electrophoresis (for study of DNA-protein binding and dissociation as well as of transcription products), nuclease protection experiments which identify base sequences covered by DNA-bound proteins, centrifugation, electron microscopy, and optical methods such as fluorescence. Transcriptional initiation at the molecular level is more complex than originally thought. The gal promoter, for instance, is known to contain overlapping start sites for mRNA synthesis, one of which responds to CAP, the other not. Recent work on this project has shown that two CAP molecules are involved in stimulation of gal transcription. Likewise, while the lac operon shows a 1:1:1 stoichiometry for CAP:polymerase:promoter interactions, there is mounting evidence that CAP and RNA polymerase may interact in more than one way at the lac control region. The proposed research involves study of the biochemistry of these systems to characterize them and to clarify how these intricate regulatory mechanisms are modulated and fine-tuned in vivo. The techniques to be used (and developed as needed) will be applicable to study of specific, non-histone chromosomal proteins in mammalian organisms. The concepts derived from this work on a bacterial system will influence future research on control processes in eukaryotic cells. Ultimately, the philosophy underlying the project is that a detailed understanding of normal, regulatory processes is crucial to unraveling of the mysteries of uncontrolled, malignant cell growth and of other pathological conditions as well.